Part of the work leading to this invention was made with U.S. Government funds. The U.S. government has certain rights in this invention.
1. Field of the Invention
The present invention relates to the fields of molecular biology and immunology. More particularly, the invention relates to the production of recombinant human thyroid peroxidase in non-thyroidal eukaryotic cells. The invention is further related to methods of using recombinant human thyroid peroxidase, and, in particular, to methods of using recombinant human thyroid peroxidase in diagnosis and treatment of immune disorders such as Hashimoto's thyroiditis.
2. Brief Description of the Related Art
Hashimoto's thyroiditis is the most common autoimmune endocrinopathy, affecting, at least subclinically, up to 15% of the adult female population (Volpe, R., In Wener's The Thyroid, 5th Edition (Ingbar, S.H., et al., Eds.), J.B. Lippincott Co., Philadelphia, pp. 1266-1291 (1986); Gordin, A., et al., Acta Endocrinol. 90:33-42 (1979)). Antibodies against a number of thyroid antigens are present in the sera of these patients, including thyroglobulin and the thyroid “microsomal” antigen (Doniach, D., et al., Clin. Endocrinol Metab. 8:63-80 (1979); Weetman, A.P., et al., Endocr. Rev. 5:309-355 (1984)). Other antigens of lesser, or uncertain, importance, include the second colloid antigen (Doniach, D., et al., Clin. Endocrinol. Metab. 8:63-80 (1979)), tubulin (Rousset, B., et al., Clin. Exp. Immunol. 52:325-332 (1983)), DNA (Katakura, M., et al., J. Clin. Endocrinol. Metab. 64:405-408 (1987)) and Autoimmune Thyroid Disease-Related Antigen I (ATRA I) (Hirayu, H., et al., J. Clin. Endocrinol. Metab. 64:578-584 (1987)).
Antibodies against the microsomal antigen, which is expressed on the cell surface (Khoury, E.L., et al., Exp. Immunol. 45:315-328 (1981); Nilsson, M., et al., Molec. Cell. Endocrinol. 55:171-185 (1987)), are believed to be of greater importance than those against thyroglobulin in the pathogenesis of Hashimoto's thyroiditis. This is because antimicrosomal antibodies (MSA) are more closely associated with the active phase of the disease (Volpe, R., In Werner's The Thyroid, 5th Edition (Ingbar, S.H., et al., Eds.), J.B. Lippincott Co., Philadelphia, pp. 1266-1291 (1986); Bogner, U., J. Clin. Endocrinol. Metab. 59:734-738 (1984); Jansson, R., et al., Clin. Exp. Immunol. 63:80-86 (1986)) and are complement-fixing (Khoury, E.L., et al., Exp. Immunol. 45:315-328 (1981)). These antibodies are, therefore, likely to initiate thyroid cellular damage.
A major recent discovery regarding Hashimoto's thyroiditis is that the previously ill-defined microsomal antigen is, at least in part, thyroid peroxidase (TPO), the primary enzyme involved in thyroid hormone synthesis. This conclusion was based on imiunologic evidence (Czarnocka, B., FEBS Letters 109:147-152 (1985); Portmann, L., et al., J. Clin. Endocrinol. Metab61:1001-1003 (1985); ; Mariotti, S., et al., J. Clin. Endocrinol. Metab. 65:987-993 (1987)) and subsequently confirmed by the molecular cloning of the cONA for these proteins (Magnusson, R.P., et al., J. Biol. Chem. 262:13885-13888 (1987); Magnusson, R.P., et al., Mol. Endocrinol. 1:856-861 (1987); Libert, R., et al., EMBO J. 6:4193-4176 (1987); Kimura, S., et al., Proc. Natl. Acad. Sci. USA 84:5555-5559 (1987)) and the discovery that their derived amino acid sequences are the same (Libert, R., et al., EMBO J. 6:4193-4176 (1987); Seto, P., et al., J. Clin. Invest. 80:1205-1208 (1987)).
Prior to the present invention, a suitable preparation of recombinant TPO has not been available for studies on the presumed abnormalities in immune regulation in Hashimoto's thyroiditis, or for the demonstration of the specific B-cell and T-cell epitopes involved in this disease. In this respect, understanding of the molecular mechanisms involved in the pathogenesis of Hashimoto's thyroiditis lags far behind that of other immune disorders, such as myasthenia gravis, a disease for which pure antigen (the acetylcholine receptor) has been obtained and epitopes already defined (Tzartos, S.J., et al., Proc. Natl. Acad. Sci. USA 85:2899-2903 (1988); Hohifeld, R., et al., J. Clin. Invest. 81:657-660 (1988)).
Human TPO (hTPO) imunopurified by monoclonal antibodies (mAbs) has been available, but is of limited value because of:                (a) inadequate supplies of human thyroid tissue; (b) the difficulties in purification of this membrane-bound antigen; and (c) contamination with other thyroid autoantigens such as thyroglobulin, which is highly abundant.        
Fragments of hTPO have been generated as recombinant bacterial (β-galactosidase) fusion proteins, and reactivity of a number of Hashimoto patient sera with small fragments of TPO expressed as fusion proteins has been reported (Libert, R., et al., EMBO J. 6:4193-4176 (1987)). Those data, however, are difficult to interpret, because the plaque assays used require extensive pre-adsorption of polyclonal antisera (Hirayu, N., et al., J. Clin. Endocrinol. Metab. 64:578-584 (1987)) and can yield false positive results.
For example, a reported fusion protein originally described as reactive with 19 of 20 Hashimoto patient sera (Libert, R., et al., EMBO J. 6:4193-4176 (1981), clone C2) has, upon immunopurification with anti-β-galactosidase mAbs, been found to react with fewer Hashimoto patient sera in an ELISA assay (Dinsart, C., et al., 17th Annual Meeting of the European Thyroid Association, Abstract #235 (1988)).
Thus, bacterial fusion proteins, too, have been of limited value because: (a) no combination of fragments has been found that reacts with all Hashimoto's sera; (b) the conformation of the fusion protein may differ from that of the native protein; and (c) the bacterial products may be toxic when added to inmune cells in culture.